Presence of SARS‐CoV‐2 virus in wastewater in the Kingdom of Bahrain during the COVID‐19 pandemic

Abstract Background Several countries, including Bahrain, used wastewater surveillance for disease activity monitoring. This study aimed to determine the presence of SARS‐CoV‐2 in untreated wastewater and to correlate it with the disease spread. Methods A retrospective review was conducted for all wastewater samples tested for SARS‐CoV‐2 in public health laboratories from November 2020 to October 2022. Samples were collected weekly between February and October 2022 from different areas across Bahrain. Real‐time polymerase chain reaction was used to test for the presence of SARS‐CoV‐2 in wastewater, and the results were correlated with the number of COVID‐19 cases in the same area. Results Of 387 wastewater samples, 103 (26.6%) samples tested positive for SARS‐CoV‐2. In late 2020, of 42 samples collected initially, four (9.5%) samples tested positive for SARS‐CoV‐2 in the four locations that hosted COVID‐19 isolation facilities. Between February and October 2022, 345 specimens of wastewater were tested, and 99 (28.7%) were positive. The highest detection rate was in February, June, and July (60%, 45%, and 43%, respectively), which corresponded to COVID‐19 peaks during 2022, and the lowest detection rate was in August and September (11% and 0%, respectively), corresponding to the low number of COVID‐19 cases. Conclusion The detection rate of SARS‐CoV‐2 in wastewater samples from Bahrain was high and was significantly correlated with the number of reported COVID‐19 cases. Wastewater surveillance can aid the existing surveillance system in monitoring SARS‐CoV‐2 spread.


| INTRODUCTION
In March 2020, the World Health Organization declared COVID-19 a pandemic. 1Since then, over 750 million cases have been detected, with more than six million deaths confirmed, making it one of the most extensive pandemics in history. 2 COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Coronaviruses are single-stranded RNA viruses belonging to the

| Sampling
Samples were collected from different residential blocks to cover all the governorates in the Kingdom of Bahrain using the grab method.
Bahrain is a small country with 778 km 2 .It is divided into four main governorates and each governorate is subdivided into residential blocks. 21On average, 10 samples were collected weekly and covered all the four governorates.A total of 387 samples from different locations were collected and analyzed, 42 samples during November and December 2020 and 345 samples during February and October 2022.
The samples were collected from a shared wastewater/sewage pipe of the residential block.The wastewater samples were collected in appropriate containers to provide enough sample volume for testing.
The amount of raw sewage collected from the selected sampling sites was 1 L per sample.Samples were kept cold (<8 C) during transportation to the laboratory for processing.The exterior surfaces of sample containers were decontaminated with 70% isopropanol and incubated in a 60 ± 1 C water bath for 1.5 h, mixed once during incubation.The water level covered the containers to reach the target temperature.
Samples were cooled to 2-8 C before proceeding with concentration.
The pasteurized sample was stored overnight at 2-8 C.

| Protocol for detection of SARS-CoV-2 from wastewater
The sample analysis method was adopted from the IDEXX reference laboratories kit in SARS-CoV-2 detection from sewage water which is a quantitative (RT-qPCR) test that can detect and quantify RNA from the SARS-CoV-2 virus in untreated wastewater. 22We followed the protocol for preparation and concentration of wastewater samples prior to nucleic acid purification and quantification of SARS-CoV-2 using IDEXX Laboratories method too. 23

| Waste water sample concentration
The sample was mixed well; then, 35 ± 1 mL was added to three empty 50 mL centrifuge tubes and centrifuged at 4700 relative centrifugal force (RCF) for 30 min at 4 ± 1 C using a swinging bucket rotor centrifuge to provide a stable bacterial pellet.Tubes were removed gently from the centrifuge bucket, and supernatant from each tube was decanted carefully into three 50 mL tubes containing 3.5 ± 0.1 g polyethylene glycol (PEG) 8000 and 0.788 ± 0.01 g sodium chloride (NaCl) and mixed at ambient temperature until completely dissolved.The liquid was decanted from each tube into three new 50 mL centrifuge tubes and centrifuged at 12,000 RCF for 120 min at 4 ± 1 C.Then, the tubes were removed from the centrifuge, and most of the supernatant was discarded from each tube carefully to avoid interfering with viral pellets.The tubes were centrifuged at 12,000 RCF for 5 min at 4 ± 1 C, then removed carefully, and the remaining supernatant from each tube was discarded; 0.4 mL nuclease-free water was transferred using pipettes to one of the tubes containing a viral pellet.The pellet was resuspended by repeatedly pipetting to rinse the inside surface of the tube around the pellet.The tube was flashed spin at 2000 ± 1000 RCF to collect all the liquid at the bottom of the tube, and then, the entire volume was transferred to the second tube containing the viral pellet after pipetting up and down several times to homogenize the concentrate.The same steps were repeated to transfer the entire volume to the third tube containing the viral pellet.Approximately 0.4 mL of the recovered concentrates were transferred to an RNase-free microtube and proceeded with nucleic acid extraction, or it could be stored overnight at À25 to À15 C.

| Real-time PCR
One milliliter of pf PCR-grade water was added to the SARS-CoV-2 mix (protected from light).Then, 200 μL of PCR-grade water was added to the positive control and incubated at 18-26 C for 10 min (both tubes), Vortex, and centrifuge (both tubes).For each sample, 10 μL SARS-CoV-2 mix and 10 μL RNA master mix were added, accounting for 10% pipette loss.The SARS-CoV-2 mix was added to empty sterile Eppendorf tubes; then, RNA master mix was added and slowly pipetted to mix.The PCR mix was vortexed, and 20 μL was added to one well per sample plus four additional wells (negative and positive PCR control and RNA extraction negative and positive control).In the first well, 5 μL of sample (purified RNA) was added; in the second well, 5 μL of positive control was added, and 5 μL of PCRgrade water (negative control) was added in the third well.Additional controls were prepared, and the plate was covered.The plate was then loaded into RT-PCR, and the cycling program was initiated, and finally, the results were analyzed and reported as positive with cycle threshold (CT) value or negatives if the CT was 38 or more.

| Data collection and analysis
The data collected include wastewater sample PCR results that were performed in public health laboratory in the Ministry of Health, samples identification number, date of sample collection, sample location (residential block and governorate), wastewater PCR results were reported as positive or negative, and CT value of positive results.The number of COVID-19 cases was reported by residential blocks, governorates, and date of testing positive.All data were entered into Microsoft Excel for analysis.Descriptive analysis of quantitative variables was done using percentages, ratios, mean, and standard deviation.For correlation tests, IBM Statistical Package for the Social Sciences (SPSS) software version 26 for statistical analysis was used.Correlation between the rate of SARS-CoV-2 positive wastewater sample detection and the reported number of COVID-19 cases in the same period was evaluated using Spearman's correlation test.
Similarly, testing for correlation between CT values of positive wastewater samples and the number of COVID-19 cases in the same area and period was done using Spearman's correlation test.P values below 0.05 were considered statistically significant.

| Detection of SARS-CoV-2 in wastewater samples
Of 387 wastewater samples collected, 103 (26.6%) samples tested positive for SARS-CoV-2.In the period between November and December 2020, a total of 42 samples were collected from wastewater drainage systems of 42 distinct residential blocks.Four (9.5%) samples tested positive for SARS-CoV-2.The positive samples were from four areas hosting COVID- 19   1).

| Correlation between the presence of the virus in the wastewater and the number of reported COVID-19 cases
Through 2022, the detection rate of positive wastewater samples was highest in February, June, and July, corresponding to 60%, 45%, and 43%, respectively.Those two periods corresponded to the two COVID-19 peaks during the year: the first peak reached a record-high number of cases in February (134,881 cases) and the second peak in (Figure 1 and Table S1).
The data were then analyzed according to the epidemiological weeks.In 2022, 31 weeks were covered by wastewater surveillance.
The number of wastewater samples tested per week ranged between 5 and 20 samples, with an average of 11 samples per week.The positivity rate was about 27% ± 22%.  2 and Table S2).

| Correlation analysis
The rate of detection of SARS-CoV-2 in wastewater samples was compared with the number of COVID-19 cases in the same epidemiological week, the number of cases the week before, and the week after using Spearman's correlation test.The rate of positive samples was significantly correlated with the reported number of COVID-19 cases.A stronger correlation was found with the reported number of cases of the same epidemiological week (r = 0.5), followed by the number of cases reported in the week after (r = 0.479) and the number of cases reported in the week before (r = 0.434).
Detected CT values were compared to the reported COVID-19 cases within the same residential blocks from where sewage samples were collected using Spearman's correlation test.The CT values detected from the 99 positive samples ranged from 30 to 37, the Lower CT values were correlated with a higher number of cases in the residential blocks from which samples were collected.

| DISCUSSION
During the active phase of COVID-19, the testing of samples was largely prompted by symptoms.People could have been shedding the virus before being symptomatic or may be asymptomatic, resulting in underdetection of the disease spread.This raises the need for a method that can help in a real-time estimation of the extent of the disease within the community to help in modulating and adjusting the pandemic response promptly. 24e wastewater-based surveillance provided an almost real-time, low-cost method to monitor the level of community transmission of SARS-CoV-2.The usefulness of wastewater surveillance had been limited by the difficulty in linking the prevalence of the virus in wastewater to the number of COVID-19 positives in the community. 25In the current study, the level of detection of COVID-19 in wastewater was correlated to the number of positive cases in the same epidemiological weeks.The correlation found between the presence of SARS-CoV-2 in wastewater and the reported number of COVID-19 cases was consistent with results from regional studies, including studies conducted in Qatar and the UAE.In Qatar, it was observed that the measured overall SARS-CoV-2 declined in wastewater with the reduction in the number of daily reported cases and an upsurge in these reported cases corresponded with an increase in the viral load detected in wastewater. 26Similarly, in the UAE, the viral load detected in wastewater diminished during the period when government-imposed measures led to a reduction in the number of recorded cases.As the daily reported cases began to increase, the viral loads detected correspondingly surged. 27The wastewater surveillance could serve as a data source and an indicator of the virus circulating within the population. 16,28e level of SARS-CoV-2 can be an indication of the presence of a cluster of cases in certain areas and can help in detecting local outbreaks.This can assist public health and hospital officials in planning for the mitigation process as well as gauge hospital bed availability and the required care needed. 29level of SARS-CoV-2 in wastewater is variable over time, and a peak of SARS-CoV-2 in wastewater increased prior to the increase in hospitalization. 24The wastewater surveillance in Bahrain was used as guidance for the COVID-19 mobile and random testing unit which was directed to the area with positive wastewater samples to help in detecting local outbreaks.In this study, no correlation was made between the increase in hospitalization and the wastewater detection of the SARS-CoV-2.
In the current study, COVID-19 cases were at par with the percentage of positive SARS-CoV-2 in wastewater during January and February 2022.Testing strategy during the pandemic included symptomatic people, asymptomatic contacts, travelers arriving in the country, and random testing done across the country for workers and in public places. 30The testing strategy was changed during March and April when travelers' testing was stopped, and testing was limited to symptomatic patients and symptomatic contacts. 28The change in the environment.SARS-CoV-2 was detected in some patients even before COVID-19 symptoms which could explain the inconsistency between the detection of the virus and COVID-related hospitalization. 32is study had some limitations.There is inconsistency in the blocks where the samples were collected.The samples were not collected from the same block each week, making following the trends of the spread of the disease difficult, but in most cases, samples were collected from the same governorate.This limitation was overcome by clumping the data of the blocks into the governorates.As Bahrain is a small country, presenting the data by governorate might be the best way of stratification and analysis.
Moreover, the wastewater surveillance was initiated in late 2020.
However, it was not conducted regularly till 2022, making it difficult to correlate all the periods where there were surges in the number of

| CONCLUSIONS
In summary, the 2020 initial testing period showed that the highest testing strategy affected the number of COVID-19-positive cases detected.Reducing the number of tests to detect COVID-19 cases may result in underestimating the true number of infections.The change in testing strategies was undertaken in most countries during the pandemic, and the reduction in COVID-19 test number may have resulted in a lower number of reported cases, thus not reflecting the actual degree of the spread of the infection.31The detection of SARS-CoV-2 in wastewater can be affected by the location, sewer shed, population size, sampling strategies, and other factors like temperature, which make the wastewater a heterogeneous F I G U R E 2 Rate of positive SARS-CoV-2 wastewater samples compared with the reported number of COVID-19 cases by epidemiological weeks of the year 2022 in the Kingdom of Bahrain.

COVID- 19
cases to the rate of wastewater detection.Despite these limitations, testing wastewater for COVID-19 acted as an indicator of the spread of the disease in the community.This study showed a correlation between SARS-CoV-2 presence in wastewater and the number of COVID cases in the community.Wastewater surveillance for SARS-CoV-2 can be used with limited resources instead of mass testing or as an adjuvant to help map the spread of the outbreak or monitor the spread.It can also be used to monitor disease activity in the long term beyond the pandemic to keep an eye on the emergence of new virus variants.
T A B L E 1 Wastewater sample tested for SARS-CoV-2 by governorate in 2022 in Bahrain.
reported COVID-19 cases had reduced to 13,947 and 16,686 cases, respectively, but the rate of positive sewage samples was noticeable, with both months recording 30% and 27%, respectively.The lowest wastewater detection rate was 11% and 0% in August and September, respectively.This period corresponded to a low number of positive COVID-19 cases, with an average of 9956 ± 1360 cases per month.October showed a similar trend, with 9371 reported COVID-19 cases but wastewater SARS-CoV-2 detection rate increased to 30% Rate of positive SARS-CoV-2 wastewater samples compared with the reported number of COVID-19 cases by month of the year 2022 in the Kingdom of Bahrain.
average CT value being 34 ± 1.6.We found a significant negative correlation of À0.45 between the CT values and COVID-19 case counts.
peak in reported COVID-19 cases.Similar trends were observed when comparing the percentage of positive wastewater samples and cases per epi-week.These findings emphasize the importance of wastewater surveillance, could be used more efficiently in future pandemics.Combined with other testing strategies, it offers a wider picture of the spread of the disease in a community compared to individual testing only.Additionally, wastewater surveillance offers valuable data guiding policymakers and helping to monitor the effectiveness of public health measures implemented.Ahmed K. Moosa: Writing-original draft; writing-review and editing.Wafa Fawzi Hasan: Formal analysis; software; validation; writing-original draft; writing-review and editing.Amjad Ghanem Mohamed: Conceptualization; data curation; formal analysis; investigation; project administration; validation; supervision; writing-review and editing.Adel Salman Al Sayyad: Conceptualization; formal analysis; methodology; validation; supervision; writing-review and editing.Maryam Y. Sanad: Data curation; investigation; writing-original draft.Maryam Alhajeri: Conceptualization.